LEF1基因在巴什拜羊不同毛色皮肤组织中的DNA甲基化及mRNA表达水平分析

doi:10.6048/j.issn.1001-4330.2022.11.018

摘要/Abstract

摘要：

【目的】分析LEF1基因在红棕色与青灰色巴什拜羊皮肤组织中DNA甲基化与mRNA的表达水平。【方法】运用BSP(亚硫酸盐修饰后测序PCR)与RT-RCR(实时荧光定量PCR),检测不同毛色巴什拜羊皮肤组织LEF1基因启动子区的甲基化水平与mRNA表达量。【结果】在红棕色巴什拜羊皮肤组织中的LEF1基因启动子区的甲基化水平高于青灰色的甲基化水平,且二者甲基化CpG位点不同,二者呈显著的负相关(P< 0.05)。【结论】DNA甲基化水平对红棕色与青灰色巴什拜羊的毛色形成具有调节作用,可作为一个候选的巴什拜羊毛色遗传标记。

关键词: 巴什拜羊; 毛色; LEF1基因; mRNA表达量; DNA甲基化

Abstract:

【Objective】 The purpose of this study is to analyze the DNA methylation and mRNA expression levels of LEF1 gene in skin tissues of red brown and blue gray Bashbay sheep. 【Methods】 The methylation level and mRNA expression of LEF1 gene promoter region in different skin tissues of Bashbay sheep were detected by BSP (sulfite Modified Sequencing PCR) and RT-RCR (real-time fluorescent quantitative PCR). 【Results】 The methylation level of LEF1 gene promoter in the skin tissue of red brown Bashbay sheep was higher than that of blue gray, and the methylation CpG sites of LEF1 gene promoter were different. At the same time, SPSS software was used to analyze the correlation between the DNA methylation level of LEF1 gene promoter and the mRNA expression, and it was found that there was a significant negative correlation between them (P< 0.05). 【Conclusion】 The results show that DNA methylation level can regulate the formation of wool color in red brown and blue gray Bashbay sheep, and they can be used as a candidate genetic marker of Bashbay sheep wool color.

Key words: Bashbay sheep; LEF1gene; mRNA expression; DNA methylation

中图分类号:

S826.8

侯晨曦, 洪文娟, 何宗龙, 决肯·阿尼瓦什. LEF1基因在巴什拜羊不同毛色皮肤组织中的DNA甲基化及mRNA表达水平分析[J]. 新疆农业科学, 2022, 59(11): 2742-2478.

HOU Chenxi, HONG Wenjuan, HE Zonglong, Jueken Aniwashi. Analysis of DNA Methylation and mRNA Expression Level of LEF1 Gene in Skin Tissues of Bashbay Sheep with Different Wool Colors[J]. Xinjiang Agricultural Sciences, 2022, 59(11): 2742-2478.

图/表 9

表1 荧光定量与BSP扩增引物

Table 1 Primers for amplicon of BSP

引物名称 Gene name	引物序列 Primer sequence	退火温度 Annealing Temp	产物大小 Product size
β- actin	F: CCAACCGTGAGAAGATGACC R: CAGAGGCGTACAGGGACAG	58	97
LEF1	F:CTCTATCCAGGCTGGTCTGC R:GGCAGCTGTCATTCTTGGAC	58	108
LEF1 -BSP	F:TATTTAGGGAAGTTAAGGTGGAG R:CTAAACTAAACTACCAAAACGACC	58	160

表2 PCR扩增反应体系

Table 2 The amplification of PCR reaction system

试剂 Reagent	体积 Volume
DNA模板Template DNA	1 μL
PCR Forward Primer	0.5 μL
PCR Reverse Primer	0.5 μL
2×Taq PCR MasterMix	13 μL
ddH2O	10 μL
总体积Total volume	25 μL

表3 PCR扩增反应程序

Table 3 The procedure of PCR amplification

程序 Program	温度 Temperature	时间 Time	循环数 Number of cycles
预变性 Pre-denaturation	94℃	5 min	-
变性 Denaturation	94℃	30s
退火 Anneal	62℃~52℃	30s	10cycle
延伸 Extension	72℃	1 min
变性 Denaturation	94℃	30s
退火 Anneal	最适温度	30s	30cycle
延伸 Extension	72℃	1 min
延伸 Extension	72℃	10 min	-
-	4℃	保存	-

图1 总RNA检测结果

Fig.1 Total RNA test results

图2 LEF1基因目的片段检测结果注:M:1 kb Plus DNA Marker;1~8:LEF1基因

Fig.2 Detection results of LEF1 gene target fragment Note: M: 1 kb Plus DNA Marker; 1~8: LEF1

图3 LEF1基因的甲基化区域预测序列

Fig.3 Methylation region prediction sequence of LEF1 gene

图4 LEF1基因甲基化区域测序峰

Fig.4 Peak sequencing of methylated region of LEF1 gene

图5 红棕色与青灰色巴什拜羊羊皮肤组织中LEF1基因的甲基化模式注:●:表示发生甲基化;○:表示未发生甲基化

Fig.5 Methylation patterns of LEF1 gene in skin tissues of red brown and bluegray bashbay sheep Note: ●: indicates methylation; ○: indicates no methylation

图6 红棕色与青灰色巴什拜羊羊皮肤组织中LEF1基因的表达量

Fig.6 Comparison of LEF1 gene expression in skin tissues of red brown and bluegray bashbay sheep

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